This invention relates to a novel DNA fragment having an active promoter region and some expression plasmids containing said DNA fragment, both of which are effective for use in the production of physiologically active substances by means of recombinant DNA technology. In particular, this invention relates to a novel DNA fragment having the promoter region of a human polypeptide chain elongation factor gene and some expression plasmids containing said DNA fragment.
With the advance of gene engineering studies, production of substances by recombinant DNA technology has come into a common means. Methods have been established almost completely for the production of foreign proteins by means of recombinant DNA technology using E. coli as the host. The use of E. coli, however, is still inappropriate for the production of a protein which essentially requires the addition of sugar chains or a protein whose physiological activity or antigenicity is altered when it is produced in a different cell type.
For the purpose of solving such a problem, various host systems have been developed using animal cells. In general, three signals are required for the gene expression in animal cells; that is, promoter, RNA splicing signal and poly (A) addition signal. It is important to select an efficient promoter for a high level expression of the gene for a protein of interest. Promoters which are being used commonly include the SV40 (a papovavirus) early promoter, adenovirus major late promoter and metallothionein promoter originated from mouse and the like. The SV40 early promoter is being used most frequently, but this promoter still has the disadvantage of low level expression capacity and narrow host range. In other words, tissue-specific expression and cell type-dependent changes in the level of expression capacity are unavoidable even if the SV40 early promoter is used. For example, the expression capacity is remarkably low in lymphoid cells and nerve cells compared to other cell types.
Recently, Y. Takebe et al. (Mol. Cell. Biol. 8:466; 1988) have constructed an SR.alpha. promoter by incorporating a portion of the terminal repeat sequence of human T-cell leukemia virus type 1 into downstream of the SV40 early promoter. According to the report, expression of the downstream gene of the SR.alpha. promoter was 1 or 2 orders of magnitude more active than that of the SV40 early promoter when a certain lymphoid cell was used as the host. However, it is still unclear whether the SR.alpha. promoter can maintain its high expression capacity in other host cells. If the diversity of useful physiologically active substances which will be produced in the future by means of recombinant DNA technology is taken into consideration, it is necessary to obtain a new promoter that has high level of expression capacity in more wider range of host cells and to develop an expression plasmid containing such a promoter.
A transient expression system, especially one in which simian COS cells are used (Gluzman, Y. 1981) Cell 23:175), is commonly employed when an attempt is made to clone a new gene by means of a biological assay using its activity as a marker. Such a transient expression system has an advantage in that a protein of interest can be obtained easily within a short period of time for use in the analysis of function and structure of the expressed protein. However, disadvantageously few expression plasmids are available for use in a transient expression system from which an expressed product can be obtained in a sufficiently large amount that the product can be detected, even by a low sensitivity biological assay system. Furthermore, until the present invention very little actually was known about an expression plasmid which could provide high expression efficiency in a broad range of host cells.